The present invention relates to a process for detecting evanescently excited luminescence with a planar dielectric optical sensor platform based on a waveguide. The invention also relates to the use of said process in qualitative affinity sensing and to the use thereof for the selective quantitative determination of luminescent components in optically turbid solutions.
When a lightwave is coupled into a planar waveguide which is surrounded by optically thinner media it is conducted by total reflectance to the interfaces of the waveguiding layer. A planar waveguide consists in the simplest case of a 3-layer system: a substrate, a wave-conducting layer, and a superstrate (e.g. the sample for assaying), the wave-conducting layer having the highest refractive index. Additional interlayers can enhance the activity of the planar waveguide still further.
A fraction of the light energy penetrates the optically thinner media. This fraction is termed the evanescent (=fading) field. The strength of the evanescent field is very strongly dependent on the thickness of the waveguiding layer itself as well as on the ratio of the refractive indices of the waveguiding layer and of the surrounding media. In the case of thin waveguides, i.e. layer thicknesses of the same or lesser thickness than of the wavelength to be guided, it is possible to distinguish discrete modes of the conducted light. With an evanescent field it is possible, for example, to excite luminescence in optically thinner media, but only directly adjacent to the guided lightwave. This principle is called evanescent luminescence excitation.
Evanescent luminescence excitation is of great interest in the analytical field, as the excitation is limited to the direct environment of the waveguiding layer.
Methods and apparatus for detecting the evanescently excited luminescence of antibodies or antigens labelled with luminescent dyes are known and described, inter alia, in U.S. Pat. No. 4,582,809. The arrangement claimed therein uses an optical fibre for the evanescent luminescence excitation. Such optical fibres typically have a diameter of up to 1 millimeter and conduct a host of modes when laser light is coupled thereinto. The evanescently excited luminescence can be measured in simple manner only by the fraction tunnelled back into the fibre. The quite large dimensions of the apparatus and the fact that comparatively large sampling volumes are required are further drawbacks. The apparatus cannot be substantially reduced in size or even miniaturised to integrated optical sensors. An enhancement of the sensitivity is usually associated with an increase in the size of the apparatus.
Photometric instruments for detecting the luminescence of biosensors under the conditions of evanescent excitation with planar optical waveguides are likewise known and disclosed, inter alia, in WO 90/06503. The waveguiding layers used therein have a thickness of 160-1000 nm, and the coupling of light into the excitation wave is effected without coupling gratings.
Various attempts have been made to enhance the sensitivity of evanescently excited luminescence and to fabricate integrated optical sensors. Thus, for example, Biosensors & Bioelectronics 6 (1991), 595-607 reports on planar monomode or low-mode waveguides which are fabricated in a two-step ion exchange process and in which the coupling of light into the excitation wave is effected with prisms. The affinity system used is human immunoglobulin G/fluorescein-labelled protein A, wherein the antibody is immobilised on the waveguide and the fluorescein-labelled protein A to be detected, in phosphate buffer, is added to a film of polyvinyl alcohol with which the measuring region of the waveguide is covered. A substantial disadvantage of this process is that only minor differences in the refraction indices between waveguiding layer and substrate layer are achievable, resulting in a relatively low sensitivity.
The sensitivity is said to be 20 nm in fluorescein isothiocyanate bonded to protein A. This is still unsatisfactory for being able to detect microtraces, and a further enhancement of sensitivity is necessary. Moreover, the reproducibility and practical viability of coupling light into the excitation wave by prisms seems difficult on account of the considerable dependence of the coupling efficiency on the quality and size of the contact area between prism and waveguide.
Another principle is proposed in U.S. Pat. No. 5,081,012. The planar waveguiding layer has a thickness of 200 nm to 1000 nm and contains two grating structures, one of which is designed as a reflection grating, so that the lightwave coupled into the waveguide must traverse the sensor region between the grating structures at least twice. Enhanced sensitivity is said to be achieved by this means. One drawback is that the reflected radiation can lead to an unwanted increase of the background radiation intensity.
The fabrication of planar wave guides is a procedure in which the planar structure of the substrate, the constant thickness and homogeneity of the waveguiding layer and the refractive index of the material used therefore are very essential. This is described, inter alia, in EP-A-0 533 074, where the proposal is made to apply inorganic waveguides to plastic substrates. This procedure has the advantage that e.g. an economically useful structuring of the coupling grating can be carried out by etching the structure into the plastics material. On the other hand, however, high demands are made of the optical quality of the plastic substrates.
Planar waveguides afford considerable advantages in large-scale production over waveguides that are based on optical fibres. In particular, it is usually necessary to provide the chopped ends of the fibres with a final polish to obtain perfect optical quality. Planar waveguides, however, can be fabricated in large dimensions and afterwards punched out, broken or cut into the desired size. Providing the edges with a final finish can in most cases be dispensed with, thereby making large-scale production more economic.
Further advantages of planar waveguides with coupling gratings are the simple calibration in the measuring device or in the measuring set-up as well as the simple application of a coating, for example to immobilise an analyte. For this purpose it is possible to use standard methods of coating technology with which reproducible constant layer thicknesses can be prepared. Typical examples of such methods are spraying, knife-coating, spin-coating or dip-coating. The quality control can likewise be carried out by known and very exact methods. Suitable methods include microscopic or interferometric methods, ellipsometry or contact angle measurements. For the curved surfaces that occcur in waveguides based on optical fibres, these methods are inapplicable or applicable only with difficulty.
Besides the actual waveguiding layer, the nature of the coupling of the lightwave into the waveguiding layer constitutes a main problem. The requirements made of gratings for coupling light into tapered waveguides for integrated optical sensors are discussed, inter alia, in Chemical, Biochemical and Environmental Fiber Sensors V, Proc. SPIE, Vol 2068, 1-13, 1994. The depth modulation of the grating and the layer thickness of the waveguide are described in this reference as crucial features. The systems proposed therein can be typically used as integrated optical light pointers, but no reference is made to a luminescence to be detected.
If it is desired to use such planar waveguides with integrated coupling gratings for luminescence measurements, then the essential features for their usefulness and for achieving a high sensitivity are a sufficiently great input-coupling efficiency, as strong an evanescent field as possible, and a low attenuation of the guided wave. These features are crucially governed by the combination of refractive index of the waveguiding layer and of the substrate material and any interlayers present, the layer thickness of the waveguide, the structure, depth modulation and grating period of the coupling grating. In addition there is the requisite optical quality of the surfaces and their planar structure or roughness.
It has now been found that it is possible, in simple manner and without an additional reflection grating, to carry out a process for the evanescent excitation and detection of luminescence with high sensitivity by combining the aforementioned crucial features such as refractive index, layer thickness and depth modulation. Typically, the attenuation of the guided lightwave is then less than 3 dB/cm, thereby resulting in a long distance of the guided beam and a low scattering of the guided wave into the media surrounding it. In particular, it is preferred under these conditions to guide the TEO and TMO mode. The conduction distance suffices, in addition to measuring the luminescence, to be able to measure with great accuracy the absorption of the excitation light in the presence of an absorbing sample.
These planar waveguides, in which only one mode or a few modes are guided, are distinguished by a particularly high sensitivity and a miniaturised construction. Normally this sensitivity is not achieved by multimode waveguides of planar or fibrous construction or, if it is achieved, then this is only possible with substantially greater geometric dimensions.
The input-coupling efficiency of the coupling grating is high, so that the intensity of the lightwave coupled into the waveguide is likewise high, resulting in conjunction with the low attenuation in an already good sensitivity.
The sensitivity is further enhanced by the evanescent field being surprisingly strong and by the high electromagnetic field strengths thereby produced contributing to a further enhancement of sensitivity. The possibility is thereby afforded of detecting even minimal amounts of luminescent material at the surface of the waveguiding layer.